Soybean is a Common Nectar Source for Honey Bees (Hymenoptera: Apidae) in a Midwestern Agricultural Landscape.

Large-scale soybean [Glycine max (L.) Merr.] cultivation has substantially transformed the Midwestern landscape in recent decades. Floral nectar produced by immense fields of soybeans has the potential to influence foraging ecology and resource accumulation of honey bee (Apis mellifera L.) colonies. In this study, we combined microscopic and molecular pollen analysis of honey samples with waggle dance inference of spatial foraging patterns to demonstrate that honey bees routinely forage on soybeans in Ohio. In analyzing honey samples from across the state, we found ubiquitous presence of soybean pollen in honey collected from agricultural lands during soybean bloom. The abundance of soybean pollen in honey increased with the amount of soybean fields surrounding the apiaries. Honey bee waggle dances recorded during soybean bloom revealed that honey bees preferred soybean fields for foraging over other habitat types. With these results, future research efforts aimed at enhancing mutual interactions between soybeans and honey bees may represent an unexplored pathway for increasing soybean production while supporting honey bees and other pollinators in the surrounding landscape.

Interspecific and intraspecific analysis of Selinum spp. collected from Indian Himalayas using DNA barcoding.

BACKGROUND: DNA barcoding is a powerful method for phylogenetic mapping and species identification. However, recent research has come to a consistent conclusion about the universality of DNA barcoding. We used matK and rbcL markers to test the universality of twelve accessions from different locations belonging to two Selinum species, Selinum tenuifolium Wall. C. B. Clarke and Selinum vaginatum C. B. Clarke, keeping in mind their ability to identify species and establish phylogenetic relationships within and between the accessions. RESULTS: The success rates of PCR amplification using matK and rbcL were 75.26% ± 3.65% and 57.24% ± 4.42%, and the rate of DNA sequencing was 63.84% ± 4.32% and 50.82% ± 4.36%, respectively, suggesting that success rates of species identification of the two fragments were higher than 41.00% (matK, 41.50% ± 2.81%; rbcL, 42.88% ± 2.59%), proving that these fragments might be used to identify species. The best evolutionary tree with good supporting values was produced utilizing combinations of matK + rbcL markers when phylogenetic relationships were built with random fragment combinations. The twelve accessions of Selinum collected from different locations and their molecular sequences of matK and rbcL markers were blasted with other genera of Apiaceae family, and it was found that Selinum is most closely related to Angelica species of Apiaceae family. CONCLUSION: The present study has grouped twelve accessions of Selinum species using molecular markers into phylogenies, which is first-of-its-kind report that established interrelationships within different species of Apiaceae with respect to Selinum.

Gene loss, genome rearrangement, and accelerated substitution rates in plastid genome of Hypericum ascyron (Hypericaceae).

BACKGROUND: Comparative genomic analysis exhibits dynamic evolution of plastid genome (plastome) in the clusioid clade of Malpighiales, which comprise five families, including multiple inversions and gene losses. Little is known about the plastome evolution in Hypericaceae, a large family in the clade. Only the plastome of one species, Cratoxylum cochinchinense, has been published. RESULTS: We generated a complete plastome sequence for Hypericum ascyron, providing the first complete plastome from the tribe Hypericeae (Hypericaceae). The H. ascyron plastome exhibits dynamic changes in gene and intron content, structure, and sequence divergence compared to the C. cochinchinense plastome from the tribe Cratoxyleae (Hypericaceae). Transcriptome data determined the evolutionary fate of the missing plastid genes infA, rps7, rps16, rpl23, and rpl32 in H. ascyron. Putative functional transfers of infA, rps7, and rpl32 were detected to the nucleus, whereas rps16 and rpl23 were substituted by nuclear-encoded homologs. The plastid rpl32 was integrated into the nuclear-encoded SODcp gene. Our findings suggested that the transferred rpl32 had undergone subfunctionalization by duplication rather than alternative splicing. The H. ascyron plastome rearrangements involved seven inversions, at least three inverted repeat (IR) boundary shifts, which generated gene relocations and duplications. Accelerated substitution rates of plastid genes were observed in the H. ascyron plastome compared with that of C. cochinchinense plastid genes. The higher substitution rates in the accD and clpP were correlated with structural change, including a large insertion of amino acids and losses of two introns, respectively. In addition, we found evidence of positive selection of the clpP, matK, and rps3 genes in the three branches related to H. ascyron. In particular, the matK gene was repeatedly under selection within the family Hypericaceae. Selective pressure in the H. ascyron matK gene was associated with the loss of trnK-UUU and relocation into the IR region. CONCLUSIONS: The Hypericum ascyron plastome sequence provides valuable information for improving the understanding of plastome evolution among the clusioid of the Malpighiales. Evidence for intracellular gene transfer from the plastid to the nucleus was detected in the nuclear transcriptome, providing insight into the evolutionary fate of plastid genes in Hypericaceae.

PCR combined with lateral flow immunochromatographic assay to differentiate the narcotic Mitragyna speciosa from related species and detect it in forensic evidence.

Plants in the genus Mitragyna (Rubiaceae) are used in traditional medicine because of their broad therapeutic activity. Four Mitragyna species, M. speciosa (Roxb.) Korth. (MS), M. rotundifolia (Roxb.) Kuntze (MR), M. diversifolia (Wall. ex G. Don) Havil. (MD), and M. hirsuta Havil. (MH), occur in Thailand. M. speciosa, commonly known as 'Kratom' in Thai, is the only narcotic species for which buying, selling, importing or possessing has been prohibited by law in Thailand and some other countries. Mitragynine and 7-hydroxymitragynine, the major psychoactive compounds, are important in the treatment of opioid withdrawal. However, this species is used in traditional medicine to relieve pain and inflammation. Consequently, a rapid and easy technique for differentiating M. speciosa from closely related species is needed for routine forensic analysis. In this study, polymerase chain reaction coupled with lateral flow immunochromatographic assay (PCR-LFA) based on matK was developed for the detection of M. speciosa in forensic specimens. Duplex primers (MS-F-FAM, Ctrl-F-DIG and Ctrl-R-Biotin) were designed based on species-specific nucleotide indels observed exclusively in the matK sequences of M. speciosa. Positive results for M. speciosa are indicated by the clear presence of three black lines on the lateral flow cassette. Forensic samples were investigated, and the three black test lines indicating M. speciosa were observed for seven of eight specimens. PCR-LFA has been proven to be fast, easy and efficient for detecting the narcotic M. speciosa and could be developed as a rapid forensic diagnostic technique for other plants.

Molecular identification and quality analysis of Rhei Rhizoma based on chloroplast matK gene and UPLC / 药学学报

Rhei Rhizoma is commonly used as a traditional Chinese medicine with multiple botanical origins. Different botanical sources may have different pharmacological activities. The germplasm resources of commercial Rhei Rhizoma were determined based on the chloroplast gene matK, and the anthraquinone and free anthraquinone content was determined by UPLC to analyze quality of commercial Rhei Rhizoma. Eighty-nine commercial Rhei Rhizoma samples were collected from 40 cities in 27 provinces. DNA was extracted and the matK gene was amplified by PCR. Results indicated that the collected samples were from the same botanical origin, Rheum palmatum, and 8 genotypes were identified, including Rp1, Rp2, Rp3, Rp4, Rp5, Rp6, Rp10 and Rp12. Rp4 and Rp6, cultivated in Gansu, Sichuan and Yunnan provinces were the main circulating genotypes, representing 40.45% and 37.08% of the total samples, respectively. Phylogenetic tree analysis showed that the eight genotypes were mainly divided into two branches, of which the main genotypes Rp4 and Rp6 were in one branch. Genetic distance analysis indicated that the genetic separation of the eight genotypes was between 0.001 and 0.010. UPLC analysis indicated that 93.26% of the samples met the Pharmacopoeia standards. There were significant differences in the content of total anthraquinone and free anthraquinone among the samples, in which the difference in free anthraquinone was 1.01% and the difference in total anthraquinone content was 3.79%, indicating that the quality of commercial Rhei Rhizoma samples varies considerably. There was no significant difference in the content of total anthraquinone and free anthraquinone in commercial Rhei Rhizoma among different collection provinces and genotypes. This study will help guide the circulation of Rhei Rhizoma in the market and provides valuable insights for molecular identification and quality analysis of other traditional Chinese medicines.

DNA barcoding of some medicinally important plant species of Lamiaceae family in India.

Several species of the Lamiaceae family are the primary source of bioactive aromatic oils and secondary metabolites, having broader applications in the cosmetics, pharmaceuticals, food, confectionery and liquor industries. Due to the scarcity of raw materials and high costs of this family's economically vital species, its products often adulterated to cater to the market's high demand. The present study provides a DNA based approach for identifying different species of this family. Henceforth, the performance of three already proposed barcode loci (matK, trnH-psbA and trnL) was examined for their PCR amplification and species recognition efficacy on various Lamiaceae species and cultivars using three different approaches such as pairwise genetic distance method, BLASTn and phylogenetic tree based on maximum likelihood (ML) analysis. Results illustrate that among all the DNA barcoding loci, matK locus can accurately and efficiently distinguish all the studied species followed by trnH-psbA and trnL. Present investigation may help diminish the illegal trade and events of adulteration of medicinally important plants species in genus Mentha, Ocimum and Plectranthus. This investigation will also help fulfil the scarcity of sequences of barcode loci of these species in the NCBI database. Apart from providing a molecular level reference for identifying processed herbal products, this technique also offers a convenient method for species identification and germplasm conservation of the Lamiaceae family.

Molecular Identification of 22 Species of Lauraceae by DNA Barcoding / 中国实验方剂学杂志

Objective:DNA barcodes suitable for Lauraceae plants were screened，and 22 Lauraceae plants were identified and classified. Method:The DNA of 22 species of Lauraceae was extracted and amplified by polymerase chain reaction（PCR） with different DNA barcoding primers，followed by electrophoresis and sequencing. Codon Code Aligner was used to proofread，splice， and remove the forward and reverse primer sequences. The sequence was imported into DNAMAN for multiple sequence alignment，and the base mutation sites were analyzed. The Kimura 2-Parameter（K2P） distance of different plants was calculated by MEGA，and the variation degree was analyzed according to the genetic distance. The phylogenetic tree was constructed based on the adjacency method. Result:Three pairs of DNA barcoding primers were used to amplify and sequence the DNA of 22 species of Lauraceae，and 20 species were identified by comparing the specific base sites of gene sequences<bold>.</bold> Conclusion:Four <italic>Litsea</italic> plants could be identified by <italic>matK</italic>， three <italic>Phoebe</italic> plants except for<italic> Cinnamomum validinerve </italic>by<italic> rbcL</italic>， and 20 Lauraceae plants by the combination of<italic> matK</italic>， <italic>rbcL</italic>， and <italic>rpoB</italic>，which provided a theoretical basis for the identification and development of Lauraceae plants. Among them，<italic>matK</italic> was predominant in the identification of Lauraceae plants，and the results also showed that the combination of sequences for plant identification could achieve a better result in DNA barcoding. This study investigated the genetic relationship between Lauraceae plants at the molecular level，aiming at providing a basis for the investigation，cultivation，development，protection， and utilization of medicinal plant resources.

DNA-Based Authentication and Metabolomics Analysis of Medicinal Plants Samples by DNA Barcoding and Ultra-High-Performance Liquid Chromatography/Triple Quadrupole Mass Spectrometry (UHPLC-MS).

There is growing interest for medicinal plants in the world drug market. Particularly, Matricaria recutita L., Valeriana officinalis L., Tilia spp., and Camellia sinensis (L.) Kuntze are some of the most consumed medicinal plants for treatment of minor health problems. Medicinal plants are seen as natural and safe; however, they can cause interactions and produce adverse reactions. Moreover, there is lack of consensus in medicinal plants regulation worldwide. DNA barcoding and UHPLC-MS technique are increasingly used to correctly identify medicinal plants and guarantee their quality and therapeutic safety. We analyzed 33 samples of valerian, linden, tea, and chamomile acquired in pharmacies, supermarkets, and herbal shops by DNA barcoding and UHPLC-MS. DNA barcoding, using matk as a barcode marker, revealed that CH1 sold as Camellia sinensis was Blepharocalyx tweediei, and sample TS2 sold as linden belong to Malvales. On the other hand, UHPLC-MS analysis revealed the presence of bioactive compounds (apigenin-7-glucoside, acetoxy valerenic acid, valerenic acid, epigallocatechin, and tiliroside). However, none of samples met minimum content of these active principles (except for valerenic acid in VF3) according to the European Medicines Agency (EMA) and Real Spanish Pharmacopeia. In conclusion, this study revealed the need to incorporate DNA barcoding and HPLC-MS techniques in quality controls of medicinal plants.

Applied Barcoding: The Practicalities of DNA Testing for Herbals.

DNA barcoding is a widely accepted technique for the identification of plant materials, and its application to the authentication of commercial medicinal plants has attracted significant attention. The incorporation of DNA-based technologies into the quality testing protocols of international pharmacopoeias represents a step-change in status, requiring the establishment of standardized, reliable and reproducible methods. The process by which this can be achieved for any herbal medicine is described, using Hypericum perforatum L. (St John's Wort) and potential adulterant Hypericum species as a case study. A range of practical issues are considered including quality control of DNA sequences from public repositories and the construction of individual curated databases, choice of DNA barcode region(s) and the identification of informative polymorphic nucleotide sequences. A decision tree informs the structure of the manuscript and provides a template to guide the development of future DNA barcode tests for herbals.

Molecular Identification and Evaluation of the Genetic Diversity of Dendrobium Species Collected in Southern Vietnam.

Dendrobium has been widely used not only as ornamental plants but also as food and medicines. The identification and evaluation of the genetic diversity of Dendrobium species support the conservation of genetic resources of endemic Dendrobium species. Uniquely identifying Dendrobium species used as medicines helps avoid misuse of medicinal herbs. However, it is challenging to identify Dendrobium species morphologically during their immature stage. Based on the DNA barcoding method, it is now possible to efficiently identify species in a shorter time. In this study, the genetic diversity of 76 Dendrobium samples from Southern Vietnam was investigated based on the ITS (Internal transcribed spacer), ITS2, matK (Maturase_K), rbcL (ribulose-bisphosphate carboxylase large subunit) and trnH-psbA (the internal space of the gene coding histidine transfer RNA (trnH) and gene coding protein D1, a polypeptide of the photosystem I reaction center (psaB)) regions. The ITS region was found to have the best identification potential. Nineteen out of 24 Dendrobium species were identified based on phylogenetic tree and Indel information of this region. Among these, seven identified species were used as medicinal herbs. The results of this research contributed to the conservation, propagation, and hybridization of indigenous Dendrobium species in Southern Vietnam.

DNA barcoding for identification of agarwood source species using trnL-trnF and matK DNA sequences.

Agarwood is a type of resinous wood found in the trunks of Aquilaria, Gonystylus, and Gyrinops species [1]. High-quality agarwood is extraordinarily expensive and therefore its source plant species have become depleted due to exploitation. In 2005, these species were added to Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [1]. Because these wild agarwood resources have become depleted, commercial production of agarwood has long been a desirable goal. In addition, inauthentic agarwood is sometimes produced from non-agarwood species. Few reports have attempted to identify source species in order to distinguish genuine from false agarwood. In this study, DNA was extracted from putative agarwood samples collected from Japanese, Indonesian, Thai, and Vietnamese markets. The trnL-trnF region and matK gene were amplified from each sample by PCR to serve as DNA barcodes for identifying the plant species to which each sample belonged. One of the wood samples did not originate from a genuine agarwood species. Although some species were identified, sequence data for agarwood source species currently available in GenBank is insufficient to identify the species to which all of these putative agarwood samples belonged. Thus, positive identification of remaining samples will require further exploration.

Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador.

BACKGROUND: Mimusops coriacea (A.DC.) Miq., (Sapotaceae), originated from Africa, were introduced to coastal areas in Ecuador where it is not extensively used as a traditional medicine to treat various human diseases. Different therapeutically uses of the species include: analgesic, antimicrobial, hypoglycemic, inflammation and pain relieve associated with bone and articulation-related diseases. Furthermore, Mimusops coriacea could be used as anti-oxidant agent. However, botanical, chemical or molecular barcode information related to this much used species is not available from Ecuador. In this study, morphological characterization was performed from leaves, stem and seeds. Furthermore, genetic characterization was performed using molecular barcodes for rbcL, matk, ITS1 and ITS2 using DNA extracted from leaves. METHODS: Macro-morphological description was performed on fresh leaves, stem and seeds. For anatomical evaluation, tissues were embedded in paraffin and transversal dissections were done following incubation with sodium hypochlorite and safranin for coloration and fixated later in glycerinated gelatin. DNA extraction was performed using a modified CTAB protocol from leaf tissues, while amplification by PCR was accomplished for the molecular barcodes rbcL, matK, ITS1 and ITS2. Sequence analysis was performed using blast in the GenBank. Phylogenetic analysis was performed with accessions queried in the GenBank belonging to the subfamily Sapotoideae. RESULTS: Leaf size was 13.56 ± 1.46 × 7.49 ± 0.65 cm; where is a macro-morphological description of the stem (see Methods). The peel of the seeds is dark brown. Sequence analysis revealed that amplicons were generated using the four barcodes selected. Phylogenetic analysis indicated that the barcodes rbcL and matK, were not discriminated between species within the same genus of the subfamily Sapotoideae. On the other hand, the ITS1 and ITS2 were discriminative at the level of genus and species of the Sapotoideae.

Employing barcoding markers to authenticate selected endangered medicinal plants traded in Indian markets.

The high demand of medicinal plants and their unrestricted collection have rendered many of these as rare or endangered. The restrictions imposed on their collection and trade are difficult to implement because of the inability to identify them in fragmented form. The rarity of these plants in nature and lack of their cultivation raise doubt about the authenticity of the herbals sold in markets. Therefore, in the present investigation, ITS/ITS2, matK, rbcL and rpoC1 sequences of fourteen species of important medicinal plants, some of which are endangered, were generated and checked for their species-specificity (sequences having maximum similarity only with their own) by BLAST1 and/or BOLD identifications. ITS sequences of 12 species were species-specific. However, ITS2 of only 10 of these 12 species were species-specific. As for the chloroplast loci, rbcL and rpoC1 sequences of all 14 species could be obtained, while matK sequences of only 10 of these could be generated. Of the retrieved sequences, rbcL, rpoC1 and matK sequences of 7, 11 and 7 species, respectively, were species-specific. The sequences of the targeted loci from the herbal samples of these species were difficult to retrieve because of failure in the amplification or sequencing. Nevertheless, based on ITS2 and/or one or more of the chloroplast loci targeted, the botanical identities of 22 herbal market samples were checked by phylogenetic tree, BLAST1 and BOLD identification methods. Of these 22 samples, only one of each of Rauvolfia serpentina and Picrorhiza kurroa were found to be authentic.

DNA barcoding of Corydalis, the most taxonomically complicated genus of Papaveraceae.

The genus Corydalis is recognized as one of the most taxonomically challenging plant taxa. It is mainly distributed in the Himalaya-Hengduan Mountains, a global biodiversity hotspot. To date, no effective solution for species discrimination and taxonomic assignment in Corydalis has been developed. In this study, five nuclear and chloroplast DNA regions, ITS, ITS2, matK, rbcL, and psbA-trnH, were preliminarily assessed based on their ability to discriminate Corydalis to eliminate inefficient regions, and the three regions showing good performance (ITS, ITS2 and matK) were then evaluated in 131 samples representing 28 species of 11 sections of four subgenera in Corydalis using three analytical methods (NJ, ML, MP tree; K2P-distance and BLAST). The results showed that the various approaches exhibit different species identification power and that BLAST shows the best performance among the tested approaches. A comparison of different barcodes indicated that among the single barcodes, ITS (65.2%) exhibited the highest identification success rate and that the combination of ITS + matK (69.6%) provided the highest species resolution among all single barcodes and their combinations. Three Pharmacopoeia-recorded medicinal plants and their materia medica were identified successfully based on the ITS and ITS2 regions. In the phylogenetic analysis, the sections Thalictrifoliae, Sophorocapnos, Racemosae, Aulacostigma, and Corydalis formed well-supported separate lineages. We thus hypothesize that the five sections should be classified as an independent subgenus and that the genus should be divided into three subgenera. In this study, DNA barcoding provided relatively high species discrimination power, indicating that it can be used for species discrimination in this taxonomically complicated genus and as a potential tool for the authentication of materia medica belonging to Corydalis.

Identification and analysis on medicinal plants of Labiatae from Emei Mountain Area Using ITS2 and matK sequences / 中草药

Objective: To evaluate the efficiency of ITS2 and matK DNA barcode for the identification the Labiatae medicinal plants in Emei Mountain. Methods: A total of 23 samples of Labiatae medicinal plants were harvested in Emei Mountain. The DNA was extracted and used for PCR to obtain the ITS2 sequences. Meanwhile, 54 ITS2 and 51 matK sequences of the medicinal plants in Labiatae were downloaded from Genbank. The interspecies and intraspecific genetic distance and sequence variation sites of all sequences were determined by MEGA 5.0 software. The Neighbor Joining (NJ) phylogenetic tree was constructed, and barcoding gap analysis was then performed. Results: The length of various ITS2 sequence was distributed from 190 to 237 bp, with GC content of 53%-73%. Moreover, significant barcoding gap was observed when comparing the distances, the recognition rate for plants of the Labiatae family was 94%.The barcoding gap of the matK sequence was not significant, there was obvious overlap, and the recognition rate for the Labiatae family was 96%. Conclusion: ITS2 has a better ability to identify Lamiaceae plants at the species level, but the matK sequence has a higher recognition rate for Lamiaceae plants. Therefore, the employment of ITS2 as core with matK as supplement was able to identify Lamiaceae plants quickly and accurately, and understand the genetic relationship between species accurately. This provides an important theoretical basis for the effective protection and rational development of the medicinal plant resources of the Labiatae plants in Emei Mountain.

[DNA barcoding identification of Dendrobium huoshanense and its adulterants].

This research preliminarily discusses the relations of Dendrobium system growth through chloroplast gene rbcL, matK and the nuclear genome ITS2. The DNA barcoding universal sequence for authentication of the Dendrobium medical plants was slected and the possibility concerning utilizing the DNA barcoding to distinguish the D. huoshanenseand its adulterants was analyzed. Using the universal primer pair of ITS2, rbcL and matK, series of extended sequencing in the Dendrobium were conducted. Meanwhile, considering the different index about amplification and sequencing success rate of each sequence, the intraspecific and interspecific aberrance, the employment of BioEdit and MEGA 5.0 software were applied to establish the systematic tree of the NJ molecular and evaluate the diversified authentication capability of various sequences. The consequence demonstrates that the sequence of ITS2 is not only the largest one both in the intraspecific and interspecific aberrance of the Dendrobium but also has obvious barcoding gap. Considering the few overlap between the intraspecific and interspecific aberrance and the highest percentage regarding the formation of unilateral branch in diverse Dendrobium which have different ITS2 sequences, it can differentiate the species of Dendrobium. Furthermore, due to the inferior success rate of the rbcL and thematK and the lower reliability of NJ systematic tree, the percentage of the unilateral species which are generated by the systematic tree of rbcL and matK sequences is deficient. Therefore, the sequence of ITS2 can serves as DNA barcoding to distinguish the D. huoshanense, the D. moniliform and the D. officinale.

Assessing universality of DNA barcoding in geographically isolated selected desert medicinal species of Fabaceae and Poaceae.

In pursuit of developing fast and accurate species-level molecular identification methods, we tested six DNA barcodes, namely ITS2, matK, rbcLa, ITS2+matK, ITS2+rbcLa, matK+rbcLa and ITS2+matK+rbcLa, for their capacity to identify frequently consumed but geographically isolated medicinal species of Fabaceae and Poaceae indigenous to the desert of Cholistan. Data were analysed by BLASTn sequence similarity, pairwise sequence divergence in TAXONDNA, and phylogenetic (neighbour-joining and maximum-likelihood trees) methods. Comparison of six barcode regions showed that ITS2 has the highest number of variable sites (209/360) for tested Fabaceae and (106/365) Poaceae species, the highest species-level identification (40%) in BLASTn procedure, distinct DNA barcoding gap, 100% correct species identification in BM and BCM functions of TAXONDNA, and clear cladding pattern with high nodal support in phylogenetic trees in both families. ITS2+matK+rbcLa followed ITS2 in its species-level identification capacity. The study was concluded with advocating the DNA barcoding as an effective tool for species identification and ITS2 as the best barcode region in identifying medicinal species of Fabaceae and Poaceae. Current research has practical implementation potential in the fields of pharmaco-vigilance, trade of medicinal plants and biodiversity conservation.

Assessment of three plastid DNA barcode markers for identification of Clinacanthus nutans (Acanthaceae).

This study was conducted to determine the feasibility of using three plastid DNA regions (matK, trnH-psbA, and rbcL) as DNA barcodes to identify the medicinal plant Clinacanthus nutans. In this study, C. nutans was collected at several different locations. Total genomic DNA was extracted, amplified by polymerase chain reaction (PCR), and sequenced using matK, trnH-psbA, and rbcL, primers. DNA sequences generated from PCR were submitted to the National Center for Biotechnology Information's (NCBI) GenBank. Identification of C. nutans was carried out using NCBI's Basic Local Alignment Search Tool (BLAST). The rbcL and trnH-psbA regions successfully identified C. nutans with sequencing rates of 100% through BLAST identification. Molecular Evolutionary Genetics Analysis (MEGA) 6.0 was used to analyze interspecific and intraspecific divergence of plastid DNA sequences. rbcL and matK exhibited the lowest average interspecific distance (0.0487 and 0.0963, respectively), whereas trnH-psbA exhibited the highest average interspecific distance (0.2029). The R package Spider revealed that trnH-psbA correctly identified Barcode of Life Data System (BOLD) 96%, best close match 79%, and near neighbor 100% of the species, compared to matK (BOLD 72%; best close match 64%; near neighbor 78%) and rbcL (BOLD 77%; best close match 62%; near neighbor 88%). These results indicate that trnH-psbA is very effective at identifying C. nutans, as it performed well in discriminating species in Acanthaceae.

DNA barcoding identification of Dendrobium huoshanense and its adulterants / 中国中药杂志

This research preliminarily discusses the relations of Dendrobium system growth through chloroplast gene rbcL, matK and the nuclear genome ITS2. The DNA barcoding universal sequence for authentication of the Dendrobium medical plants was slected and the possibility concerning utilizing the DNA barcoding to distinguish the D. huoshanenseand its adulterants was analyzed. Using the universal primer pair of ITS2, rbcL and matK, series of extended sequencing in the Dendrobium were conducted. Meanwhile, considering the different index about amplification and sequencing success rate of each sequence, the intraspecific and interspecific aberrance, the employment of BioEdit and MEGA 5.0 software were applied to establish the systematic tree of the NJ molecular and evaluate the diversified authentication capability of various sequences. The consequence demonstrates that the sequence of ITS2 is not only the largest one both in the intraspecific and interspecific aberrance of the Dendrobium but also has obvious barcoding gap. Considering the few overlap between the intraspecific and interspecific aberrance and the highest percentage regarding the formation of unilateral branch in diverse Dendrobium which have different ITS2 sequences, it can differentiate the species of Dendrobium. Furthermore, due to the inferior success rate of the rbcL and thematK and the lower reliability of NJ systematic tree, the percentage of the unilateral species which are generated by the systematic tree of rbcL and matK sequences is deficient. Therefore, the sequence of ITS2 can serves as DNA barcoding to distinguish the D. huoshanense, the D. moniliform and the D. officinale.

Evaluating five different loci (rbcL, rpoB, rpoC1, matK, and ITS) for DNA barcoding of Indian orchids.

Orchidaceae, one of the largest families of angiosperms, is represented in India by 1600 species distributed in diverse habitats. Orchids are in high demand owing to their beautiful flowers and therapeutic properties. Overexploitation and habitat destruction have made many orchid species endangered. In the absence of effective identification methods, illicit trade of orchids continues unabated. Considering DNA barcoding as a potential identification tool, species discrimination capability of five loci, ITS, matK, rbcL, rpoB, and rpoC1, was tested in 393 accessions of 94 Indian orchid species belonging to 47 genera, including one listed in Appendix I of CITES and 26 medicinal species. ITS provided the highest species discrimination rate of 94.9%. While, among the chloroplast loci, matK provided the highest species discrimination rate of 85.7%. None of the tested loci individually discriminated 100% of the species. Therefore, multi-locus combinations of up to five loci were tested for their species resolution capability. Among two-locus combinations, the maximum species resolution (86.7%) was provided by ITS+matK. ITS and matK sequences of the medicinal orchids were species specific, thus providing unique molecular identification tags for their identification and detection. These observations emphasize the need for the inclusion of ITS in the core barcode for plants, whenever required and available.